Purpose:
Usher syndrome type III (USH3A) is an autosomal recessive disorder caused by mutations in the Clarin-1 gene resulting in sensorineural hearing loss and progressive retinal degeneration. We previously showed that a high titer of AAV Clarin-1 vector (1uL of 8.43x10 12 vg/ml) delivered to the retina is toxic and this toxicity is reduced by using lower doses. In the present work, we used a GRK photoreceptor cell specific promoter in an AAV2 quadruple capsid mutant (Y272F+Y444F+ Y500F+Y730F) (AAV2 quad) vector to avoid Clarin-1 overexpression toxicity. We also employed several commercial anti-clarin antibodies to evaluate the location of both the endogenous and the AAV-delivered Clarin-1.

Methods:
Commercial antibodies were acquired for anti-human Clarin-1, paraffin section staining was optimized in C57BL/6 mouse control retinas, and analyzed in Clarin-1 knockout mice. An AAV2 quad vector was then used to deliver diluted Clarin-1 subretinally (SR) and the same AAV2 quad vector with an additional T491V mutation was used to target photoreceptors after an intravitreal (IV) vector delivery. Both vectors contain a GRK photoreceptor specific promoter driving human CLRN1 with a hemagglutinin (HA) tag at its C-terminus. Mice were injected with 1 uL SR at a 1:100 (2.01 x1010 vg/ml) dilution or 1 uL IV at a full titer (2.31 x1012 vg/ml) into wild-type C57BL/6 mice and Clarin-1 knockout mice. Retinas were analyzed by electroretinography (ERG), Optical Coherence Tomography (OCT), and histology using anti-HA and anti-Clarin-1 antibodies.

Results:
Antibody staining showed Clarin-1 localization in the connecting cilium (CC) of photoreceptor cells, which supports previously published data. Clarin-1 antibodies also co-localized with gamma-tubulin, a basal body marker for the CC. No AAV Clarin-1 toxicity was observed by OCT or ERG upon either SR delivery of 1:100 diluted vector or undiluted IV delivered vector.

Conclusions:
Endogenous Clarin-1 appears to be localized to the CC in photoreceptor cells, where it co-localizes with connecting cilium markers and confirms that photoreceptors are a viable target cell for gene treatment of Ush3A. There appeared to be no toxicity associated with either GRK AAV construct. Overall, this data suggests that Clarin-1 may play a role in photoreceptor cells like many other Usher proteins and additionally suggests an optimal AAV vector design for Ush3A gene therapy.